Ebola hemorrhagic fever is among the deadliest of communicable diseases. Its causative agent, Ebola virus, is categorized as both an emerging infectious disease, a Class A biological agent with potential for use in bioterrorism, and a Select Agent. Despite these rather dramatic facts, there are currently no available treatments for people suffering with this disease. This discrepancy between need and supply is in large part due to our lack of understanding of the basic biology of Ebola virus. Ebola virus is classified in the family Filoviridae. It consists of a single, approximately 19-kilobase strand of negative-sense RNA that is tightly wrapped in viral proteins. Formation of this ribonucleoprotein complex, referred to as the nucleocapsid, is hypothesized to commence with interactions between the viral nucleocapsid protein and the 5' termini of the replicating viral RNA. The specific mechanisms that guide this interaction are unknown. This application begins to address the long-term goal of understanding the overall mechanism of encapsidation in Ebola virus. Specifically, residues within the viral RNA and the nucleocapsid protein that facilitate nucleocapsid formation will be identified. An NP-RNA binding assay will be established to measure the dissociation constant for this interaction. Preliminary truncations of the RNA and the protein will establish regions of each that are necessary for binding. Cross-linking experiments will also be utilized for this analysis. Directed mutagenesis will then isolate residues within the RNA and the protein that are intricately involved in nucleocapsid formation. The results of these in vitro studies will be tested in reconstituted viral replication systems. Both mini-replication systems and a full viral reverse genetics system, in which the entire virus is reconstituted from plasmids, are available for these experiments. The outcome from this research will be significant as it will fill a gap in our knowledge of the replication of Ebola virus and thus lead to the future development of antiviral therapies aimed at inhibition of encapsidation, an essential, but poorly understood step in the lifecycle of Ebola virus.